EPA Method 1603 “Spike The Ball”

Debmalya Bhattacharyya Ph.D (DEB)

Northeast Ohio Regional Sewer District

EPA Method 1603 • Escherichia coli (E. coli) in Water by Membrane Filtration

Using Modified membrane-Thermotolerant Escherichia coli Agar (Modified mTEC)

• The minimum analytical QC requirements for the analysis of samples using Method 1603 to demonstrate laboratory capability (Section 9.0) • IPR initial precision and recovery (4 spikes into PBS) • OPR ongoing precision and recovery (Every 20 samples or

once a week) • MS Matrix Spike (Every 20 samples or once a week or a new

source) • Positive and negative controls • Filter sterility checks • Method blanks • Media sterility checks

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QC Acceptance Criteria 1603

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QC Sample Requirements

BioBall • A BioBallTM is a small water

soluble ball containing a precise number of microorganisms that is manufactured and is certified for quality control by the company

• BioBall™ 550 • BioBall™ 1*10e8

Laboratory Prepared • A lab-prepared culture is

used to spike samples • E. coli ATCC #11775 is

cultured in Tryptic Soy Broth (TSB)

• Concentration is validated by analyzing serial dilutions of the culture

• Must validate the culture concentration with every spike analyzed

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Initial Method Validation (IPR) • Validation with the BioBall™

– Certified concentration with COA – No matrix interference when using PBS or Saline – No need to maintain frozen cultures

• Problems encountered – Low recovery on mTEC agar at 35°C/44.5°C

• Performed multiple analyses – mTEC at 35°C using PBS and Saline – mTEC at 35°C/44.5°C in PBS and Saline – Colilert™ – PCA at 35°C in PBS and Saline

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Initial Test Results

Method/Agar SolutionIncubationTemp. °C

Theoretical Value (CFU)

Actual Value (CFU) % Recovery

Colilert BioBall PBS 35 550 326 59%Colilert BioBall Saline 35 500 308 62%Colilert BioBall Saline 35 50 32 64%mTEC BioBall PBS 35 55 4.4 8%mTEC BioBall Saline 35 50 9 18%mTEC BioBall PBS 35/44.5 55 4.2 8%mTEC BioBall Saline 35/44.5 50 13 26%PCA BioBall Saline 35 50 46 92%

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Manufacturer Recommendations

• Manufacturer stated that recoveries will be decreased using;

• Membrane filtration • Selective media • Temperature other than 35°C • Using PBS instead of Saline • These decreased recoveries still method

requirements (detect – 144%)

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Alternate Spiking Method

• Grow E. coli #11775 into log phase (4-5 hours) in TSB • Performed multiple analyses • ATCC states 11775 is thermo-tolerant as sited in EPA

Method 1603 and 1103.1 – Other thermo-tolerant strains available

• ATCC 43894 and ATCC 25922

• Analyzed by different methods • mTEC at 35°C using PBS • mTEC at 35°C/44.5°C in PBS • mFC at 35°C using PBS • mFC at 35°C/44.5°C in PBS • Colilert™

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ATCC #11775 on mTEC

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Low % Recovery Altered colony morphology

Spiking with an environmental strain

• Decided to culture an environmental strain of E. coli from wastewater treatment plant effluent – Multiple colonies pooled grown at 44.5°C – Multiple passages grown at 44.5°C

• Identification confirmed by MALDI-TOF – Escherichia coli ATCC 25922 THL

• The PBS was then spiked with the environmental strain (30 cfu/100 ml).

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Success with environmental strain

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Sample CFUE. coli spike 35°C #1 31E. coli spike 35°C #2 22E. coli spike 35°C #3 28E. coli spike 35°C #4 27E. coli spike 35°C #5 32

Average 28

Sample CFUE. coli spike 35°C #1 33E. coli spike 35°C #2 31E. coli spike 35°C #3 27E. coli spike 35°C #4 28E. coli spike 35°C #5 34

Average 31

35°C Environmental E. coli spike

35°C Environmental E. coli spike

Colilert

PCA: Spread Plate

Comparable % recoveries observed in all methods

1603 93%

103%

87%

100%

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Very good and consistent % recoveries. The colony morphologies under both conditions were comparable.

NEORSD Implementation

• NEORSD Prepared Spike using a thermo-tolerant environmental strain – Cultured a strain from one of our typical

environmental samples – Prepare aliquots to use throughout the year – Perform validation of the prepared aliquots – Freeze aliquots at -80°C to reduce culturing time and

continuous validation of the spikes – Perform analysis of frozen aliquots to verify viability – Environmental strain had best recoveries via EPA

1603

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2015 QC Summary 2015 QC Summary

NEORSD Published NEORSD PublishedAverage %R 94% 93%

Precision %RSD 20% 36% 32%Control Limits 57 - 130 38 - 127 33 - 153 12 - 149

OPR Matrix Spike

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2015 IPR Worksheet

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Conclusion • The additional required QC (IPR, OPR and MS) in

EPA method 1603 and EPA 1600 are beneficial and recoveries can be obtained using procedures defined in the method – There are difficulty using organisms from dry sources

like Bioballs, thus resulting in lower recoveries – The ATCC 1175 provides better recoveries than

BioballsTM

• Lab cultures provide better recovery however can be difficult to maintain

• Utilizing a true thermo tolerant strain provides the best recovery and reproducibility

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Future Direction

• Experiment with an ATCC thermotolerant strain suchas ATCC 43894 and ATCC 25922

• Incorporate the OPR, IPR and MS QC requirementswith environmental samples

• Utilize these QC procedures with the Colilertmethod

• Utilize IPR and OPR with other microbial methods– Similar to chemical analysis IDOC and ODOC

» Initial Demonstration of Capabilities» Ongoing Demonstration of Capabilities

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Questions ?

• Contact Information Mark Citriglia Northeast Ohio Regional Sewer District citrigliam@neorsd.org

– Special Thanks to:

• Nichole Schafer • Christopher Lannan • Rose Read

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QC Definitions • Section 9.0 Quality Control

– Initial Precision and Recovery (IPR) – demonstrate acceptable method performance is performed using a known strain of E. coli or Enterococci

– Ongoing Precision and Recovery (OPR) – demonstrate ongoing control of the analytical system is performed using a known strain of E. coli or Enterococci

• Every 20 samples or once a week

– Matrix Spike (MS) – determine the organisms recovery in a specific matrix (disinfected wastewater samples)

• Every 20 samples or once a week or a new source

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Problem Identification

• Performed multiple analyses• mTEC at 35°C using PBS and Saline• mTEC at 35°C/44.5°C in PBS and Saline• Colilert™

• PCA at 35°C in PBS and Saline

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